烟草乙烯转录因子ERF基因NtTOE3的克隆、载体构建及表达分析

Cloning,Construction of Expression Vector and Expression Analysis of the Ethylene-responsive Factor ERF Gene NtTOE3 in Nicotiana tabacum

ERF(Ethylene-responsive factor)是一类具有AP2特征结构域的乙烯应答转录因子,在响应植物的逆境胁迫应答中起关键作用。为明确ERF家族成员TOE3在烟草中的生物学功能和调控机制,同源克隆普通烟草K326中NtTOE3基因cDNA全长,利用实时荧光定量PCR(qRT-PCR)表征基因表达模式,结合生物信息学分析,对基因功能和调控机制进行初步预测。结果表明,该基因全长1 356 bp,编码451个氨基酸残基,预测分子量为49. 84 ku。生物信息学分析表明,该蛋白是一个亲水蛋白,可能定位在细胞核,且与美花烟草(Nicotiana sylvestris) NsTOE3有96%的同源性,故命名为NtTOE3。组织表达分析发现,该基因在烟草根、茎、叶、花中均有表达,其中茎中表达量最高。逆境胁迫试验表明,该基因能快速响应低钾、高盐、干旱、H_2O_2、ABA和4℃等逆境处理。表明NtTOE3转录因子在烟草非生物胁迫中起着重要调节作用。

ERF（Ethylene-responsive factor） is a class of ethylene-responsive transcription factor with AP2 signature domain, which plays a pivotal role in response to stress response in plants.To identify the biological functions and regulation mechanism of ERF familymember TOE3 in tobacco k326, one NtTOE3 gene swerecloned from Nicotiana tabacum with homologous cloning strategy, gene expression patterns were analyzed byreal-time quantitative PCR（qRT-PCR）, and their functions and regulation mechanisms were predicted bybioinformatic analysis.Theresultsshowed that NtTOE3 contained 1 356 bp and encoded 451 amino acid. The predicted molecular weight was 49.84 ku. By bioinformatics analysis, NtTOE3 was a hydrophilic protein which might be localized in the nucleus,andthe protein had 96% homology with N.sylvestris NsTOE3, thusit was namedas NtTOE3 . From expression patterns, the gene was expressed in roots, stems, leaves and flowers in mature stage, which had the highest expression in stems. Expression patterns under abiotic stress indicated the gene responded rapidly to the adverse conditions of low potassium, high salt, drought, H 2O 2, ABA and 4℃. Therefore, the NtTOE3 transcription factor plays an important regulatory role in tobacco abiotic stress.